Pores In Cell Membrane Research Articles

Cavitation bubble dynamics in microfluidic gaps of variable height

We study experimentally the dynamics of laser-induced cavitation bubbles created inside a narrow gap. The gap height, h , is varied from 15 to 400 microm and the resulting bubble dynamics is compared to a semiunbounded fluid. The cavitation bubbles are created with pulsed laser light at constant laser energy and are imaged with a high-speed camera. The bubble lifetime increases with decreasing gap height by up to 50% whereas the maximum projected bubble radius remains constant. Comparing the radial dynamics to potential flow models, we find that with smaller gaps, the bubble-induced flow becomes essentially planar, thus slower flows with reduced shear. These findings might have important consequences for microfluidic applications where it is desirable to tune the strength and range of the interactions such as in the case of cell lysis and cell membrane poration.

Arginine-Rich Peptides Destabilize the Plasma Membrane, Consistent with a Pore Formation Translocation Mechanism of Cell-Penetrating Peptides

Recent molecular-dynamics simulations have suggested that the arginine-rich HIV Tat peptides translocate by destabilizing and inducing transient pores in phospholipid bilayers. In this pathway for peptide translocation, Arg residues play a fundamental role not only in the binding of the peptide to the surface of the membrane, but also in the destabilization and nucleation of transient pores across the bilayer. Here we present a molecular-dynamics simulation of a peptide composed of nine Args (Arg-9) that shows that this peptide follows the same translocation pathway previously found for the Tat peptide. We test experimentally the hypothesis that transient pores open by measuring ionic currents across phospholipid bilayers and cell membranes through the pores induced by Arg-9 peptides. We find that Arg-9 peptides, in the presence of an electrostatic potential gradient, induce ionic currents across planar phospholipid bilayers, as well as in cultured osteosarcoma cells and human smooth muscle cells. Our results suggest that the mechanism of action of Arg-9 peptides involves the creation of transient pores in lipid bilayers and cell membranes.

Cholesterol-dependent cytolysins induce rapid release of mature IL-1β from murine macrophages in a NLRP3 inflammasome and cathepsin B-dependent manner

CDC are exotoxins secreted by many Gram-positive bacteria that bind cholesterol and oligomerize to form pores in eukaryotic cell membranes. We demonstrate that CDC TLO induces caspase-1 cleavage and the rapid release of IL-1beta from LPS-primed murine BMDM. IL-1beta secretion depends on functional toxin pore formation, as free cholesterol, which prevents TLO binding to cell membranes, blocks the cytokine release. Secretion of the mature forms of IL-1beta and caspase-1 occurs only at lower TLO doses, whereas at a higher concentration, cells release the biologically inactive proforms. IL-1beta release at a low TLO dose requires potassium efflux, calcium influx, and the activities of calcium-independent PLA(2), caspase-1, and cathepsin B. Additionally, mature IL-1beta release induced by a low TLO dose is dependent on the NLRP3 inflammasome, and pro-IL-1beta release induced by a high TLO dose occurs independently of NLRP3. These results further elucidate a mechanism of CDC-induced IL-1beta release and suggest a novel, immune evasion strategy in which IL-1beta-containing macrophages might release primarily inactive cytokine following exposure to high doses of these toxins.

Glycosylphosphatidylinositols are potential targets for the development of novel inhibitors for aerolysin‐type of pore‐forming bacterial toxins

Many bacteria produce toxins that cause damage through the formation of pores in the host cell membrane. Some of these toxins, such as aerolysin, use glycosylphosphatidylinositols (GPIs) as their binding receptors to assist the pore formation on the host cell surface and the subsequent insertion of the resultant pores into the cell membrane. GPIs are a class of complex glycolipids that anchor surface proteins and glycoproteins onto the cell membrane in eukaryotic species. This review has summarized the reported evidences supporting the GPI-dependent pore-forming mechanism for aerolysin-type of toxins and analyzed the possibility of targeting this unique process for the design and development of novel GPI-based inhibitors for these pore-forming bacterial toxins.

Requirements for the Formation of Membrane Pores by the Reovirus Myristoylated μ1N Peptide

The outer capsid of the nonenveloped mammalian reovirus contains 200 trimers of the micro1 protein, each complexed with three copies of the protector protein sigma3. Conformational changes in micro1 following the proteolytic removal of sigma3 lead to release of the myristoylated N-terminal cleavage fragment micro1N and ultimately to membrane penetration. The micro1N fragment forms pores in red blood cell (RBC) membranes. In this report, we describe the interaction of recombinant micro1 trimers and synthetic micro1N peptides with both RBCs and liposomes. The micro1 trimer mediates hemolysis and liposome disruption under conditions that promote the micro1 conformational change, and mutations that inhibit micro1 conformational change in the context of intact virus particles also prevent liposome disruption by particle-free micro1 trimer. Autolytic cleavage to form micro1N is required for hemolysis but not for liposome disruption. Pretreatment of RBCs with proteases rescues hemolysis activity, suggesting that micro1N cleavage is not required when steric barriers are removed. Synthetic myristoylated micro1N peptide forms size-selective pores in liposomes, as measured by fluorescence dequenching of labeled dextrans of different sizes. Addition of a C-terminal solubility tag to the peptide does not affect activity, but sequence substitution V13N or L36D reduces liposome disruption. These substitutions are in regions of alternating hydrophobic residues. Their locations, the presence of an N-terminal myristoyl group, and the full activity of a C-terminally extended peptide, along with circular dichroism data that indicate prevalence of beta-strand secondary structure, suggest a model in which micro1N beta-hairpins assemble in the membrane to form a beta-barrel pore.

Fish venom: pharmacological features and biological significance

AbstractNearly 1200 species of marine fish are venomous and they account for two‐third of the population of venomous vertebrates. Fish venoms are focused as a potential source of pharmacological agents and physiological tools that have evolved to target vital processes in the human body that appear to have more electivity than many drugs. Fish venoms possess cardiovascular, neuromuscular, oedematic and cytolytic activity. Lethal toxins have been isolated and purified, with some having LD50 values comparable to that of snake venoms. Cardiovascular activity seems to be the dominant effect of fish venoms in experimental models. Piscine venom acts both pre‐ and post‐junctionally to produce depolarization of cell membranes. Studies on cytolytic activity of fish venom found that it produces lysis by forming hydrophilic pores in cell membranes which then result in cell lysis. Almost all fish venoms with neuromuscular activity also possesses cytolytic activity, and it is very likely that the two activities are related. Fish venom is known to induce intense and sustained edematogenic response. As piscine venoms have evolved for the same purpose, they show a number of similarities pharmacologically and it seems likely that most of the biological activities of any given toxin can be traced back to its cytolytic activity. A variety of toxins have been isolated from piscine venom. Although there is a complex balance between the components present in the venom of different fish, all of them seem to share similar activity – functionally and pharmacologically as well as structurally.

Interaction of single biological cell with tandem microbubbles in microfluidics.

Coupled oscillation of two laser generated microbubbles (maximum radius = 28 μm) and associated shear stresses are investigated experimentally. Bubble‐bubble interaction in a microchannel of 25 μm height is observed using high‐speed video cameras and μPIV technique. Two liquid microjets moving in opposite directions can be generated when the second bubble is produced at the maximum size of the first one. The interaction of these tandem microbubbles with single cell leads to controllable poration of adjacent cell membrane and dye uptake. μPIV data are compared with cell viability at various bubble‐cell distances and azimuthal orientations. This method provides a new approach for highly selective cell treatment in situ, applicable to targeted microinjection of macromolecules and gene vectors in microfluidics devices. [Work supported in part by NIH.]

The P2X7Receptor Drives Microglial Activation and Proliferation: A Trophic Role for P2X7R Pore

Microglial activation is an integral part of neuroinflammation associated with many neurodegenerative conditions. Interestingly, a number of neurodegenerative conditions exhibit enhanced P2X(7) receptor (P2X(7)R) expression in the neuroinflammatory foci where activated microglia are a coexisting feature. Whether P2X(7)R overexpression is driving microglial activation or, conversely, P2X(7)R overexpression is a consequence of microglial activation is not known. We report that overexpression alone of a purinergic P2X(7)R, in the absence of pathological insults, is sufficient to drive the activation and proliferation of microglia in rat primary hippocampal cultures. The trophic responses observed in microglia were found to be P2X(7)R specific as the P2X(7)R antagonist, oxidized ATP (oxATP), was effective in markedly attenuating microgliosis. oxATP treatment of primary hippocampal cultures expressing exogenous P2X(7)Rs resulted in a significant decrease in the number of activated microglia. P2X(7)R is unusual in exhibiting two conductance states, a cation channel and a plasma membrane pore, and there are no pharmacological agents capable of cleanly discriminating between these two states. We used a point mutant of P2X(7)R (P2X7RG345Y) with intact channel function but ablated pore-forming capacity to establish that the trophic effects of increased P2X(7)R expression are exclusively mediated by the pore conductance. Collectively, and contrary to previous reports describing P2X(7)R as a "death receptor," we provide evidence for a novel trophic role for P2X(7)R pore in microglia.

Pore formation byVibrio choleraecytolysin follows the same archetypical mode as β‐barrel toxins from gram‐positive organisms

Vibrio cholerae cytolysin (VCC) forms SDS-stable heptameric beta-barrel transmembrane pores in mammalian cell membranes. In contrast to structurally related pore formers of gram-positive organisms, no oligomeric prepore stage of assembly has been detected to date. In the present study, disulfide bonds were engineered to tie the pore-forming amino acid sequence to adjacent domains. In their nonreduced form, mutants were able to bind to rabbit erythrocytes and to native erythrocyte membranes suspended in PBS solution and form SDS-labile oligomers. These remained nonfunctional and represented the long-sought VCC prepores. Disulfide bond reduction in these oligomers released the pore-forming sequence from its locked position, and subsequent membrane insertion led to formation of SDS-stable pores and hemolysis. Addition of increasing amounts of an inactive mutant to wild-type toxin resulted in the formation of mixed oligomers with progressively reduced SDS stability on membranes. Membrane insertion of active monomers in these hybrid oligomers was still observed, but the functional pore diameter was reduced. These findings indicate that formation of an oligomeric prepore precedes membrane insertion of the pore-forming amino acid sequence and demonstrate that pore formation by VCC follows the same archetypical pathway as beta-barrel cytolysins of gram-positive organisms such as staphylococcal alpha-toxin.

Ultrasound Exposure of Lipoplex Loaded Microbubbles Facilitates Direct Cytoplasmic Entry of the Lipoplexes

Recently we reported that the transfection of cells by PEGylated lipoplexes becomes significantly better by binding the PEGylated lipoplexes to the surface of microbubbles and applying ultrasound. To further optimize this gene delivery system it is important to understand the working mechanism. This paper elucidates the cellular entry path of these lipoplexes. The results clearly show that the PEGylated lipoplexes, released from the microbubbles upon applying ultrasound, are not taken up by endocytosis, the most common route for nanoparticles to enter cells. Our data demonstrate that, upon implosion of the microbubbles, the PEGylated lipoplexes are released and are most probably able to passively diffuse through the cell membrane pores or become injected in the cytoplasm of the target cells. This is attractive as the in vivo use of PEGylated nanoparticles remains currently limited due to a decreased cellular uptake and inefficient escape of the PEGylated nanoparticles from the endosomes.

Poisson-Nernst-Planck Models of Nonequilibrium Ion Electrodiffusion through a Protegrin Transmembrane Pore

Protegrin peptides are potent antimicrobial agents believed to act against a variety of pathogens by forming nonselective transmembrane pores in the bacterial cell membrane. We have employed 3D Poisson-Nernst-Planck (PNP) calculations to determine the steady-state ion conduction characteristics of such pores at applied voltages in the range of −100 to +100 mV in 0.1 M KCl bath solutions. We have tested a variety of pore structures extracted from molecular dynamics (MD) simulations based on an experimentally proposed octomeric pore structure. The computed single-channel conductance values were in the range of 290–680 pS. Better agreement with the experimental range of 40–360 pS was obtained using structures from the last 40 ns of the MD simulation, where conductance values range from 280 to 430 pS. We observed no significant variation of the conductance with applied voltage in any of the structures that we tested, suggesting that the voltage dependence observed experimentally is a result of voltage-dependent channel formation rather than an inherent feature of the open pore structure. We have found the pore to be highly selective for anions, with anionic to cationic current ratios (ICl−/IK+) on the order of 103. This is consistent with the highly cationic nature of the pore but surprisingly in disagreement with the experimental finding of only slight anionic selectivity. We have additionally tested the sensitivity of our PNP model to several parameters and found the ion diffusion coefficients to have a significant influence on conductance characteristics. The best agreement with experimental data was obtained using a diffusion coefficient for each ion set to 10% of the bulk literature value everywhere inside the channel, a scaling used by several other studies employing PNP calculations. Overall, this work presents a useful link between previous work focused on the structure of protegrin pores and experimental efforts aimed at investigating their conductance characteristics.

Micro-electroporation of mesenchymal stem cells with alternating electrical current pulses

Micro-electroporation is an electroporation technology in which the electrical field that induces cell membrane poration is focused onto a single cell contained in a micro-electromechanical structure. Micro-electroporation has many unique attributes including that it facilitates real time control over the process of electroporation at the single cell level. Flow-through micro-electroporation expands on this principle and was developed to facilitate electroporation of a large numbers of cells with control over the electroporation of every single cell. However, our studies show that when electroporation employs conventional direct current (DC) electrical pulses the micro-electroporation system fails, because of electrolysis induced gas bubble formation. We report in this study that when certain alternating currents (AC) electrical pulses are used for micro-electroporation it becomes possible to avoid electrolytic gas bubble formation in a micro-electroporation flow-through system. The effect of AC micro-electroporation on electrolysis was found to depend on the AC frequency used. This concept was tested with mesenchymal stem cells and preliminary results show successful electroporation using this system.

Experimental study on cell self-sealing during sonoporation

Reparable sonoporation of human breast cancer cells was achieved during exposure to moderate ultrasound (spatial peak acoustic pressure, p sp = 0.25 MPa, 1 MHz tone-bursts, 20 cycles per tone-burst at pulse repetition frequency of 10 kHz) up to 40 s assisted by the presence of encapsulated microbubbles (EMBs). It was demonstrated that shear stress generated by oscillating EMBs at the cell membranes introduced small transient pores in cell membranes by which cells were able to uptake some extracellular fluid and meanwhile triggered the repairing process through self-sealing during sonoporation. It was also indicated by post-sonoporation analysis using the fluorescent microscopy, scanning electron microscopy, and the Bradford assay which determined the protein content in cell supernatant that the self-sealing might be established by lysosomal-associated membrane protein, LAMP-1, fusing with the plasma membrane under the stressful condition in sonoporation.

Model Assessment of Cell Membrane Breakdown in Clusters and Tissues Under High-Intensity Electric Pulsing

This paper presents a simulation study of cell membrane electroporation in clusters by high-intensity voltage pulses. The focus is on assessing effects associated with: 1) the variability in shape and randomness of the cells within clusters; 2) the density of clusters; 3) the effects in heterogeneous tissues; 4) the role of pulse width on fractional electroporation for given electrical characteristics; and 5) conductivity and cell shape influences on the electric strength versus pulse duration behavior. Quantitative results are obtained based on two-dimensional, time-dependent, random Voronoi network analyses. The calculations predict that it is harder to electroporate cells in a cluster due to the random orientation of cell boundaries with regard to the applied field. Also, with increasing cellular distortions and shape irregularity, the poration is predicted to require higher voltage amplitudes or longer pulse durations to cause the same effects. Intracellular conductivity was shown to be a primary parameter influencing cell membrane poration, with membrane permittivity having a secondary effect. This has implications for tissue selectivity, especially for ultrashort duration pulsing. Finally, strength-duration (S-D) curves have been obtained, and shown to depend on the relative disorder and randomness within clusters.

The C-terminus of IpaC is required for effector activities related to Shigella invasion of host cells

Invasion plasmid antigen C (IpaC) is secreted by the Shigella flexneri type III secretion system (TTSS) as an essential trigger of epithelial cell invasion. At the molecular level, IpaC possesses a distinct functional organization. The IpaC C-terminal region between amino acids 319 and 345 is predicted to form a coiled-coil structure. Such alpha-helical motifs appear to be a recurring structural theme among TTSS components. Together with IpaB, this IpaC region is also required for the formation of translocon pores in target cell membranes. In contrast, mutations within the C-terminal tail of IpaC (defined by residues 345–363) have no effect on contact hemolysis (a putative measure of translocon pore formation), but they can contribute significantly to IpaC's ability to trigger S. flexneri entry into cultured cells. Here we describe the molecular dissection of the IpaC C-terminus and how changes in this region affect selected virulence-related activities. IpaC invasion function requires its immediate C-terminus and this general region may be involved in its ability to trigger actin nucleation. In contrast, IpaC could not be shown to interact directly with Cdc42, a host GTPase closely tied to Shigella invasion.

3H]A-804598 ([3H]2-cyano-1-[(1S)-1-phenylethyl]-3-quinolin-5-ylguanidine) is a novel, potent, and selective antagonist radioligand for P2X7 receptors

ATP-sensitive P2X7 receptors are localized on cells of immunological origin including peripheral macrophages and glial cells in the CNS. Activation of P2X7 receptors leads to rapid changes in intracellular calcium concentrations, release of the pro-inflammatory cytokine IL-1β, and following prolonged agonist exposure, the formation of cytolytic pores in plasma membranes. Data from gene knockout studies and recently described selective antagonists indicate a role for P2X7 receptor activation in inflammation and pain. While several species selective P2X7 antagonists exist, A-804598 represents a structurally novel, competitive, and selective antagonist that has equivalent high affinity at rat (IC50=10nM), mouse (IC50=9nM) and human (IC50=11nM) P2X7 receptors. A-804598 also potently blocked agonist stimulated release of IL-1β and Yo-Pro uptake from differentiated THP-1 cells that natively express human P2X7 receptors. A-804598 was tritiated ([3H]A-804598; 8.1Ci/mmol) and utilized to study recombinant rat P2X7 receptors expressed in 1321N1 cells. [3H]A-804598 labeled a single class of high affinity binding sites (Kd=2.4nM and apparent Bmax=0.56pmol/mg). No specific binding was observed in untransfected 1321N1 cells. The pharmacological profile for P2X antagonists to inhibit [3H]A-804598 binding correlated with their ability to block functional activation of P2X7 receptors (r=0.95, P<0.05). These data demonstrate that A-804598 is one of the most potent and selective antagonists for mammalian P2X7 receptors described to date and [3H]A-804598 is a high affinity antagonist radioligand that specifically labels rat P2X7 receptors.

Structural analysis of the essential self-cleaving type III secretion proteins EscU and SpaS

During infection by Gram-negative pathogenic bacteria, the type III secretion system (T3SS) is assembled to allow for the direct transmission of bacterial virulence effectors into the host cell. The T3SS system is characterized by a series of prominent multi-component rings in the inner and outer bacterial membranes, as well as a translocation pore in the host cell membrane. These are all connected by a series of polymerized tubes that act as the direct conduit for the T3SS proteins to pass through to the host cell. During assembly of the T3SS, as well as the evolutionarily related flagellar apparatus, a post-translational cleavage event within the inner membrane proteins EscU/FlhB is required to promote a secretion-competent state. These proteins have long been proposed to act as a part of a molecular switch, which would regulate the appropriate chronological secretion of the various T3SS apparatus components during assembly and subsequently the transported virulence effectors. Here we show that a surface type II beta-turn in the Escherichia coli protein EscU undergoes auto-cleavage by a mechanism involving cyclization of a strictly conserved asparagine residue. Structural and in vivo analysis of point and deletion mutations illustrates the subtle conformational effects of auto-cleavage in modulating the molecular features of a highly conserved surface region of EscU, a potential point of interaction with other T3SS components at the inner membrane. In addition, this work provides new structural insight into the distinct conformational requirements for a large class of self-cleaving reactions involving asparagine cyclization.

Community-acquired methicillin-resistant Staphylococcus aureus pneumonia

Community-acquired methicillin-resistant Staphylococcus aureus is increasingly recognized as an important pathogen causing skin and soft tissue infections. We report a case of severe necrotizing pneumonia caused by community-acquired methicillin-resistant S. aureus in a peripartum woman. This case illustrates that community-acquired methicillin-resistant S. aureus must be considered as a potential pathogen in severe community-acquired pneumonia.

Inactivation of host Akt/protein kinase B signaling by bacterial pore-forming toxins.

Uropathogenic Escherichia coli (UPEC) are the major cause of urinary tract infections (UTIs), and they have the capacity to induce the death and exfoliation of target uroepithelial cells. This process can be facilitated by the pore-forming toxin alpha-hemolysin (HlyA), which is expressed and secreted by many UPEC isolates. Here, we demonstrate that HlyA can potently inhibit activation of Akt (protein kinase B), a key regulator of host cell survival, inflammatory responses, proliferation, and metabolism. HlyA ablates Akt activation via an extracellular calcium-dependent, potassium-independent process requiring HlyA insertion into the host plasma membrane and subsequent pore formation. Inhibitor studies indicate that Akt inactivation by HlyA involves aberrant stimulation of host protein phosphatases. We found that two other bacterial pore-forming toxins (aerolysin from Aeromonas species and alpha-toxin from Staphylococcus aureus) can also markedly attenuate Akt activation in a dose-dependent manner. These data suggest a novel mechanism by which sublytic concentrations of HlyA and other pore-forming toxins can modulate host cell survival and inflammatory pathways during the course of a bacterial infection.

P2Z purinoceptors.

In response to tetra-anionic ATP4-, P2Z receptors signal opening of a non-selective plasma membrane pore which permits passage across cell membranes of ions, nucleotides and other small molecules that are usually membrane impermeant. P2Z receptor-induced pores on murine macrophages, macrophage-like cell lines and human culture-matured macrophages are permeable to molecules of up to 831 Da. The function of P2Z receptors is unknown. Also unknown is whether the binding site for ATP4- and the transmembrane pore, the properties that characterize P2Z receptors, reside on a single protein or reflect the activities of two or more proteins. That ATP(4-) -unresponsive cell lines do not express connexin 43 has led Beyer and Steinberg to suggest that opening or surface expression of this gap junction protein is induced by P2Z receptors. Xenopus oocytes, injected with cRNA transcribed from a pool of 100 cDNA clones isolated from a murine macrophage-derived cDNA library, and treated with ATP4-, express a non-selective membrane conductance characteristic of P2Z receptors. The conductance induced with cRNA is smaller than that induced by mRNA from macrophages, suggesting the presence of a dominant subunit of a multicomponent receptor in this pool of 100 cDNA clones.